The culturing of cells is a highly complex matter as different cell types demand different types of liquid medium as well as different growth conditions in order to obtain optimal growth of the cells. The growth conditions include chemical composition and flow rate of the medium, mechanical stimulation, and electromagnetic stimulation.
Cells can be cultured in 2D (dimensional) layers and have traditionally been cultured in culture tissue flasks and culture plates. In this manner, the cells are grown in a monolayer where the liquid medium is added on top of the cells. The culture flasks and dishes are placed inside an incubator in order to optimise the temperature and CO2 level. However, monolayer cultures are not optimal for cells as they do not experience conditions similar to their natural environment. In order to obtain a more natural environment for the cells, changes in the growth conditions can be induced as for example, changes of the oxygen level.
It has been shown through numerous experiments that in most cases 3D cell cultures mimic the in vivo situation much closer than 2D cell cultures, especially concerning primary cells. The main reason is that the natural environment typically is 3D. Therefore, 3D cell cultures represent an important field for modelling/controlling the complex biological processes in vitro There is a big difference between a flat layer of cells and a complex, 3D tissue (Abbott A, “Biology's new dimension”, Nature 21:870-872, (2003). For example, in 2D cultures, both normal and malignant mammary epithelial cells have similar, high levels of Coxsackievirus and adenovirus receptors (CAR). But in 3D cultures, only malignant cells have an upregulation of CAR (Anders M et al. Proc. Natl. Acad. Sci. USA 100, 1943-1948, (2003).
Furthermore, cell culture experiments with embryonic stem (ES) cell proliferation and differentiation in 3D scaffolds also show a greater cell proliferation and differentiation than 2D cultures (Willerth S M, et al., “Optimization of fibrin scaffolds for differentiation of murine embryonic stem cells into neural lineage cells”, Biomaterials, 27:5990-6003, (2006).
For adult stem cells such as human mesenchymal stem cells (hMSCs), 3D culturing has proven to be superior to 2D conventional culturing in relation to the osteogenic potential of stem cells in vitro (Machado C B et al., “3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells”, Biomed. Mater. 2:124-131, (2007); Grayson W L et al., “Human mesenchymal stem cells tissue development in 3D PET matrices”, Biotechnol Prog., 20(3):905-12, (2004); 3D Culturing is Superior to 2D Conventional Culturing in Examining The Osteogenic Potential of Stem Cells In Vitro, 3D Biotek, LLC, North Brunswick, N.J., 675 US Highway 1, North Brunswick, N.J. 08902, http://3dbiotek.com/Documents/3DScaffold_Osteogenesis.pdf.).
Even if the differentiation is successful in 2D the usage in clinical applications has been limited, because the architecture of the formed extracellular matrix is diverse from the native tissue morphology.
In order to obtain proper differentiated cells which can be used for tissue engineering purposes, different 3D culturing processes with the use of porous scaffolds have been developed.
3D cultures require means of increasing the flow of nutrients and oxygen to the cells and removal of waste products from the cells situated centrally in the scaffold, as simple diffusion is insufficient for transport at distances longer than approx. 200 μm (Ko HCH et al., “Engineering thick tissues—the vascularisation problem”, European Cells and Materials, 14:1-19, (2007).
Sufficient transport to the centre of the scaffold can be achieved by spinning the cells in flasks—so called spinner flasks—as described for example in EP 1 736 536 A2. The cells are adherent to scaffolds which are then arranged in spinner flasks filled with liquid medium. The medium is set in motion relative to the scaffolds with a magnetic stirrer bar or a shafted impeller to provide a convective means to enhance nutrient/waste exchange to and from the fixed scaffold. This fluid motion effects increased shear on the adherent cells, which is known to influence cell differentiation.
The main drawback of this culture method is that the scaffolds are not thoroughly or evenly perfused. Furthermore, because the viscous flow field around each scaffold is dependent on the exact spatial position in the flask, it is difficult to achieve consistent results when culturing more than 8 samples in one flask. This is a disadvantage of this method, as it increases the overall footprint of the perfusion setup.
The increased mass transport due to convection is limited to a volume near the surface of the scaffolds. The interior of the scaffold is still reliant on diffusion. As for the effects of increased shear stress on the differentiation of the cells, these are also confined to the cells located superficially in the scaffold.
Other methods comprises perfusion flow where small scaffolds can be situated at the bottom of culture racks and liquid medium is directed across the scaffolds in order to supply the nutrients in a continuous manner (Cartmell S H et al., “Effects of Medium Perfusion Rate on Cell-Seeded Three-Dimensional Bone Constructs in Vitro”,. Tissue Engineering, 9(6):1197-1203, (2003); Bancroft G N et al., “Technical Note: Design of a Flow Perfusion Bioreactor System for Bone Tissue-Engineering Applications”, Tissue Engineering, 9(3):549-554, (2003)). However, these methods have huge drawbacks. For the perfusion flow, the equipment itself is not ideal since a large amount of tubes are needed in order to sustain a constant flow of liquid medium. Furthermore, a large amount of equipment like pumps and flasks are arranged inside the incubator, thus taking up large amounts of valuable incubator shelf space.
Another method of perfusing scaffolds is to mount the scaffolds on a micro-controlled linearly actuated plunger, which then moves reciprocally up and down within a medium containing vessel (Timmins N E et al., “Three-Dimensional Cell Culture and Tissue Engineering in a T-CUP (Tissue Culture Under Perfusion)”, Tissue Engineering, 13(8):2021-2028, (2007). This system fails to eliminate the need for tubing and comprises a large number of assembly parts. Furthermore, although the mean flow through the scaffold can be calculated, non-uniformity between individual scaffolds will lead to non-uniform perfusion.